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OPEN
received: 26 August 2015 accepted: 03 May 2016 Published: 02 June 2016
Bone scaffolds loaded with siRNASemaphorin4d for the treatment of osteoporosis related bone defects Yufeng Zhang1,2, Lingfei Wei1, Richard J. Miron1,3,4, Bin Shi1,2 & Zhuan Bian1 Osteoporosis is a prominent disorder affecting over 200 million people worldwide. Recently, semaphorins have been implicated in the cell-cell communication between osteoclasts and osteoblasts and have been associated with the progression of osteoporosis. Previously, we demonstrated that knockdown of semaphorin4d (Sema4d) using siRNA delivered with a bone-targeting system prevented bone loss in an osteoporotic animal model. Here, we used this bone-specific technology containing siRNA-Sema4d and fabricated a PLLA scaffold capable of enhancing bone repair following fracture. We investigated the ability of the implant to release siRNA-Sema4d into the surrounding tissues over time and to influence new bone formation in a 3 mm femur osteoporotic defect model in ovariectomized rats. Delivery of the bone-targeting system released from PLLA scaffolds began 2 hours post-implantation, peaked at 1 day, and was sustained over a 21 day period. μCT analysis demonstrated a significantly higher bone volume/total volume bone mineral density and number of osteoblasts in the rats that were transplanted with scaffolds loaded with siRNA-Sema4d. These results confirm the specific role of Sema4d in bone remodeling and demonstrate that significant increases in the speed and quality of new bone formation occur when siRNA-Sema4d is delivered via a PLLA scaffold. Osteoporosis is a global healthcare issue with an increasing socio-economic burden. It is caused by an imbalance between bone-forming osteoblasts and bone-resorbing osteoclasts1,2. Over 200 million people are affected worldwide3, with the majority of patients being white or Asian women over 65 years old4. For decades, research has shown that osteoporotic patients demonstrate reduced healing after bone injury5. Fractures are more common, and their healing potential is drastically reduced5. The disease is caused by hyper-activity of osteoclasts, which affects the bone remodeling cycle and limits the ability of the incoming bone-forming osteoblasts to lay new bone matrix2,6–10. At present, the two major pharmacological approaches for the treatment of osteoporosis are as follows: stimulation of bone formation via anabolic agents (such as parathyroid hormone) or prevention of bone resorption via anti-resorptives (such as bisphosphonates, calcitonin, raloxifene, and estrogen replacement therapy)11. Semaphorins have recently been targeted as molecules with osteoporosis treatment potential. They are directly implicated in the cell-cell communication between osteoclasts and osteoblasts and may be a novel target for the treatment of osteoporosis12–21. Furthermore, the overexpression of semaphorin4d (Sema4d) in bone tissues has been associated with osteoporosis in an animal model22. A Sema4d knockout animal model recently demonstrated an increase in bone thickness and density, further implicating Sema4d in the bone remodeling cycle. Previously, we have developed a site-specific bone-targeting drug delivery system consisting of polymeric nanoparticles containing an siRNA-mediated gene knockdown system for Sema4d23. This system specifically targets native bone and releases siRNA-Sema4d onto bone surfaces occupied by osteoclasts. Weekly injections of this system significantly improved bone formation in both an early and late phase osteoporotic animal model by re-balancing the bone remodeling cycle23. While most of the current osteoporosis research focuses on fracture prevention using a variety of pharmacological agents, the treatment of osteoporosis-related defects following fracture has not been as well studied. 1
State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, People’s Republic of China. 2Department of Dental Implantology, School and Hospital of Stomatology, Wuhan University, People’s Republic of China. 3Department of Restorative, Preventive and Pediatric Dentistry, University of Bern, Switzerland. 4 Department of Periodontology, Nova Southeastern University, Fort Lauderdale, Florida, USA. Correspondence and requests for materials should be addressed to Z.B. (email: [email protected]) Scientific Reports | 6:26925 | DOI: 10.1038/srep26925
1
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Figure 1. The kinetics of Asp8-(STR-R8) release/distribution in femurs over 21 days. The epi-fluorescence indicated sustained and localized drug distribution restricted to the defect and the adjacent bone tissue and peaking at 1 day post-implantation.
Therefore, in this study, we fabricated a specific bone replacement material from poly-L-lactic acid (PLLA) scaffolds to promote bone formation in an osteoporotic phenotype and studied this in 3 mm femur defects in ovariectomized (OVX) rats. The PLLA scaffolds were then loaded with a bone-specific targeting system that contained siRNA-Sema4d to improve bone remodeling, and new bone formation was investigated.
Materials and Methods
Preparation and characterization of PLLA. Four groups were used for all animal experiments: 1) drilled
control, 2) PLLA alone, 3) PLLA-(Asp8-(STR-R8)) and 4) PLLA-(Asp8-(STR-R8)-siRNA-Sema4d. Porous PLLA scaffolds were prepared using a previously described freeze-drying method24. Briefly, 1g PLLA (Sigma #765112; Sigma-Aldrich, St. Louis, USA) was dispersed in 10 ml dioxane (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) under vigorous stirring until completely dissolved. Then, the mixture was rapidly transferred to a freezer at −35 °C overnight to solidify the solvent and induce solid–liquid phase separation. This solidified mixture was then maintained at −80 °C for 2 h and subsequently transferred into a freeze-drying vessel (Christ Beta 1–15, Germany) for 48 h until dried. Scaffolds were then sterilized and lyophilized. The Asp8-(STR-R8)-siRNASema4d (covalently linked) bone targeting system was fabricated as previously described23. Briefly, the packaging of siRNA (GenePharma, Suzhou, China) was performed according to a protocol adapted from DNA transfection experiments25. Asp8-Stearyl-R8 (1.5 μl) (ChinaPeptides Co., Ltd, Shanghai, China) was diluted in 50 μl of unsupplemented Neurobasal medium (Gibco , USA) and combined with 10 pmol of siRNA in 50 μL of unsupplemented Neurobasal medium. The solution was incubated for 5 min at room temperature. The PLLA (0.01 g) was then incubated with 1 ml of Asp8-(STR-R8)-siRNA-Sema4d solution or Asp8-(STR-R8) solution (5 OD/ml) at 4 °C overnight to allow complete infiltration. These complexes were then frozen by immersion at −80 °C for 2 h and subsequently lyophilized.
®
Animals and surgical procedures. Mature female Wistar rats (12 weeks old, mean body weight 250 g) were purchased and used for this study. All handling and surgical procedures were approved by the Ethics Committee for Animal Research, Wuhan University, China. The methods were carried out in “accordance” with the approved guidelines. Animals received food and water ad libitum and were housed at a constant temperature of 22 °C. For surgery, the animals were place under general anesthesia using an intraperitoneal injection of chloral hydrate (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China, 10%, 4 ml/kg body weight), and all operations were performed under sterile conditions with a minimally invasive surgical technique. Postoperatively, penicillin Scientific Reports | 6:26925 | DOI: 10.1038/srep26925
2
www.nature.com/scientificreports/
Figure 2. Representative images of overall bone regeneration using 3D reconstruction at 8 weeks postimplantation. The siRNA-Sema4d treated group had significantly increased mineralization in trabecular bone compared to the control, PLLA alone and PLLA-(Asp8-(STR-R8)-siRNA-Sema4d loaded defects. The bottom row represents the 3-dimensional reconstruction of the bone defects from the micro-CT analysis, demonstrating higher bone density for the group treated with siRNA-Sema4d. (40,000 IU/ml, 1 ml/kg) was injected each day for 3 days. There were no signs of inflammation or other notable anomalies.
Osteoporosis model. The animals were acclimatized to the new laboratory surroundings for one week. The osteoporotic animal model was established using a bilateral ovariectomy (OVX)26. Briefly, under general anesthesia, the rats received 10 mm linear incisions bilaterally in the lumbar skin. Both ovaries were gently removed. The tissue layers were then repositioned and sutured. Following surgery, buprenorphine (0.05 mg/kg) was administered by subcutaneous injection for pain management. Femoral defect model. Standardized 3 mm femoral cylindrical defects were created in ovariectomized ani-
mals 2 months after the bilateral removal of the ovaries. The osteoporosis model should have been established at this time point according to previous studies27,28. A distal femoral epiphysis linear skin incision (approximately 1 cm) was performed bilaterally. The femoral condyles were then exposed by blunt dissection of the muscles surrounding the femurs. A 2.8 mm diameter reamer was used to create a 3.0 mm diameter anteroposterior bicortical channel perpendicular to the shaft axis (Supplemental Fig. 1A (reprint with permission from Cheng et al.27)). The bur was irrigated at a slow speed (800 rpm) and rinsed with saline solution to avoid thermal necrosis of the cells and tissues. The bone fragments were washed out of the cavity using the saline solution. Scaffolds (3 mm in diameter by 5 mm in thickness) were gently placed bilaterally according to a randomized group allocation. The incisions were then closed. Buprenorphine (0.05 mg/kg) was administered by subcutaneous injection for postoperative pain management.
Pharmacokinetics study. PLLA scaffolds were cut using custom trays (3 mm in diameter and 5 mm in
thickness), gently loaded with rhodamine-labeled Asp8-(STR-R8) and placed within the femurs. Samples were collected at 0 hours, 12 hours, 1 day, 3 days, 7 days, 14 days and 21 days post-operation to assess the duration and location of drug distribution. The fluorescence signal was detected using a Xenogen IVIS imaging system (Xenogen, Alameda, Ca, USA) as previously described23.
Bone regeneration. To study bone regeneration in the osteoporotic femoral defect, the rats were divided into four groups: 1) drilled control, 2) filled with PLLA, 3) filled with PLLA-(Asp8-(STR-R8)) and 4) filled with PLLA-(Asp8-(STR-R8)-siRNA-Sema4d) (n = 15 in all groups). Rats were sacrificed at 2, 4 and 8 weeks after femoral surgery. The defect, size, shape and location are presented in supplemental Fig. 1. The rats were divided into four groups with 6 defects per group per time point.
Micro-CT (μCT) analysis of osteogenesis in the femoral defect. After harvesting at each time point,
the femurs were removed intact and fixed in 4% freshly prepared formaldehyde for 24 h at 4 °C. The μCT imaging system (μCT50, Scanco Medical, Brüttisellen, Switzerland) was used to evaluate the osteogenesis within the
Scientific Reports | 6:26925 | DOI: 10.1038/srep26925
3
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Figure 3. Statistical analysis of bone volume/total volume (BV/TV), trabecular thickness and tissue mineral density in the bone defect as assessed by μCT. All data are shown as the mean ± SD. P
OPEN
received: 26 August 2015 accepted: 03 May 2016 Published: 02 June 2016
Bone scaffolds loaded with siRNASemaphorin4d for the treatment of osteoporosis related bone defects Yufeng Zhang1,2, Lingfei Wei1, Richard J. Miron1,3,4, Bin Shi1,2 & Zhuan Bian1 Osteoporosis is a prominent disorder affecting over 200 million people worldwide. Recently, semaphorins have been implicated in the cell-cell communication between osteoclasts and osteoblasts and have been associated with the progression of osteoporosis. Previously, we demonstrated that knockdown of semaphorin4d (Sema4d) using siRNA delivered with a bone-targeting system prevented bone loss in an osteoporotic animal model. Here, we used this bone-specific technology containing siRNA-Sema4d and fabricated a PLLA scaffold capable of enhancing bone repair following fracture. We investigated the ability of the implant to release siRNA-Sema4d into the surrounding tissues over time and to influence new bone formation in a 3 mm femur osteoporotic defect model in ovariectomized rats. Delivery of the bone-targeting system released from PLLA scaffolds began 2 hours post-implantation, peaked at 1 day, and was sustained over a 21 day period. μCT analysis demonstrated a significantly higher bone volume/total volume bone mineral density and number of osteoblasts in the rats that were transplanted with scaffolds loaded with siRNA-Sema4d. These results confirm the specific role of Sema4d in bone remodeling and demonstrate that significant increases in the speed and quality of new bone formation occur when siRNA-Sema4d is delivered via a PLLA scaffold. Osteoporosis is a global healthcare issue with an increasing socio-economic burden. It is caused by an imbalance between bone-forming osteoblasts and bone-resorbing osteoclasts1,2. Over 200 million people are affected worldwide3, with the majority of patients being white or Asian women over 65 years old4. For decades, research has shown that osteoporotic patients demonstrate reduced healing after bone injury5. Fractures are more common, and their healing potential is drastically reduced5. The disease is caused by hyper-activity of osteoclasts, which affects the bone remodeling cycle and limits the ability of the incoming bone-forming osteoblasts to lay new bone matrix2,6–10. At present, the two major pharmacological approaches for the treatment of osteoporosis are as follows: stimulation of bone formation via anabolic agents (such as parathyroid hormone) or prevention of bone resorption via anti-resorptives (such as bisphosphonates, calcitonin, raloxifene, and estrogen replacement therapy)11. Semaphorins have recently been targeted as molecules with osteoporosis treatment potential. They are directly implicated in the cell-cell communication between osteoclasts and osteoblasts and may be a novel target for the treatment of osteoporosis12–21. Furthermore, the overexpression of semaphorin4d (Sema4d) in bone tissues has been associated with osteoporosis in an animal model22. A Sema4d knockout animal model recently demonstrated an increase in bone thickness and density, further implicating Sema4d in the bone remodeling cycle. Previously, we have developed a site-specific bone-targeting drug delivery system consisting of polymeric nanoparticles containing an siRNA-mediated gene knockdown system for Sema4d23. This system specifically targets native bone and releases siRNA-Sema4d onto bone surfaces occupied by osteoclasts. Weekly injections of this system significantly improved bone formation in both an early and late phase osteoporotic animal model by re-balancing the bone remodeling cycle23. While most of the current osteoporosis research focuses on fracture prevention using a variety of pharmacological agents, the treatment of osteoporosis-related defects following fracture has not been as well studied. 1
State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, People’s Republic of China. 2Department of Dental Implantology, School and Hospital of Stomatology, Wuhan University, People’s Republic of China. 3Department of Restorative, Preventive and Pediatric Dentistry, University of Bern, Switzerland. 4 Department of Periodontology, Nova Southeastern University, Fort Lauderdale, Florida, USA. Correspondence and requests for materials should be addressed to Z.B. (email: [email protected]) Scientific Reports | 6:26925 | DOI: 10.1038/srep26925
1
www.nature.com/scientificreports/
Figure 1. The kinetics of Asp8-(STR-R8) release/distribution in femurs over 21 days. The epi-fluorescence indicated sustained and localized drug distribution restricted to the defect and the adjacent bone tissue and peaking at 1 day post-implantation.
Therefore, in this study, we fabricated a specific bone replacement material from poly-L-lactic acid (PLLA) scaffolds to promote bone formation in an osteoporotic phenotype and studied this in 3 mm femur defects in ovariectomized (OVX) rats. The PLLA scaffolds were then loaded with a bone-specific targeting system that contained siRNA-Sema4d to improve bone remodeling, and new bone formation was investigated.
Materials and Methods
Preparation and characterization of PLLA. Four groups were used for all animal experiments: 1) drilled
control, 2) PLLA alone, 3) PLLA-(Asp8-(STR-R8)) and 4) PLLA-(Asp8-(STR-R8)-siRNA-Sema4d. Porous PLLA scaffolds were prepared using a previously described freeze-drying method24. Briefly, 1g PLLA (Sigma #765112; Sigma-Aldrich, St. Louis, USA) was dispersed in 10 ml dioxane (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) under vigorous stirring until completely dissolved. Then, the mixture was rapidly transferred to a freezer at −35 °C overnight to solidify the solvent and induce solid–liquid phase separation. This solidified mixture was then maintained at −80 °C for 2 h and subsequently transferred into a freeze-drying vessel (Christ Beta 1–15, Germany) for 48 h until dried. Scaffolds were then sterilized and lyophilized. The Asp8-(STR-R8)-siRNASema4d (covalently linked) bone targeting system was fabricated as previously described23. Briefly, the packaging of siRNA (GenePharma, Suzhou, China) was performed according to a protocol adapted from DNA transfection experiments25. Asp8-Stearyl-R8 (1.5 μl) (ChinaPeptides Co., Ltd, Shanghai, China) was diluted in 50 μl of unsupplemented Neurobasal medium (Gibco , USA) and combined with 10 pmol of siRNA in 50 μL of unsupplemented Neurobasal medium. The solution was incubated for 5 min at room temperature. The PLLA (0.01 g) was then incubated with 1 ml of Asp8-(STR-R8)-siRNA-Sema4d solution or Asp8-(STR-R8) solution (5 OD/ml) at 4 °C overnight to allow complete infiltration. These complexes were then frozen by immersion at −80 °C for 2 h and subsequently lyophilized.
®
Animals and surgical procedures. Mature female Wistar rats (12 weeks old, mean body weight 250 g) were purchased and used for this study. All handling and surgical procedures were approved by the Ethics Committee for Animal Research, Wuhan University, China. The methods were carried out in “accordance” with the approved guidelines. Animals received food and water ad libitum and were housed at a constant temperature of 22 °C. For surgery, the animals were place under general anesthesia using an intraperitoneal injection of chloral hydrate (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China, 10%, 4 ml/kg body weight), and all operations were performed under sterile conditions with a minimally invasive surgical technique. Postoperatively, penicillin Scientific Reports | 6:26925 | DOI: 10.1038/srep26925
2
www.nature.com/scientificreports/
Figure 2. Representative images of overall bone regeneration using 3D reconstruction at 8 weeks postimplantation. The siRNA-Sema4d treated group had significantly increased mineralization in trabecular bone compared to the control, PLLA alone and PLLA-(Asp8-(STR-R8)-siRNA-Sema4d loaded defects. The bottom row represents the 3-dimensional reconstruction of the bone defects from the micro-CT analysis, demonstrating higher bone density for the group treated with siRNA-Sema4d. (40,000 IU/ml, 1 ml/kg) was injected each day for 3 days. There were no signs of inflammation or other notable anomalies.
Osteoporosis model. The animals were acclimatized to the new laboratory surroundings for one week. The osteoporotic animal model was established using a bilateral ovariectomy (OVX)26. Briefly, under general anesthesia, the rats received 10 mm linear incisions bilaterally in the lumbar skin. Both ovaries were gently removed. The tissue layers were then repositioned and sutured. Following surgery, buprenorphine (0.05 mg/kg) was administered by subcutaneous injection for pain management. Femoral defect model. Standardized 3 mm femoral cylindrical defects were created in ovariectomized ani-
mals 2 months after the bilateral removal of the ovaries. The osteoporosis model should have been established at this time point according to previous studies27,28. A distal femoral epiphysis linear skin incision (approximately 1 cm) was performed bilaterally. The femoral condyles were then exposed by blunt dissection of the muscles surrounding the femurs. A 2.8 mm diameter reamer was used to create a 3.0 mm diameter anteroposterior bicortical channel perpendicular to the shaft axis (Supplemental Fig. 1A (reprint with permission from Cheng et al.27)). The bur was irrigated at a slow speed (800 rpm) and rinsed with saline solution to avoid thermal necrosis of the cells and tissues. The bone fragments were washed out of the cavity using the saline solution. Scaffolds (3 mm in diameter by 5 mm in thickness) were gently placed bilaterally according to a randomized group allocation. The incisions were then closed. Buprenorphine (0.05 mg/kg) was administered by subcutaneous injection for postoperative pain management.
Pharmacokinetics study. PLLA scaffolds were cut using custom trays (3 mm in diameter and 5 mm in
thickness), gently loaded with rhodamine-labeled Asp8-(STR-R8) and placed within the femurs. Samples were collected at 0 hours, 12 hours, 1 day, 3 days, 7 days, 14 days and 21 days post-operation to assess the duration and location of drug distribution. The fluorescence signal was detected using a Xenogen IVIS imaging system (Xenogen, Alameda, Ca, USA) as previously described23.
Bone regeneration. To study bone regeneration in the osteoporotic femoral defect, the rats were divided into four groups: 1) drilled control, 2) filled with PLLA, 3) filled with PLLA-(Asp8-(STR-R8)) and 4) filled with PLLA-(Asp8-(STR-R8)-siRNA-Sema4d) (n = 15 in all groups). Rats were sacrificed at 2, 4 and 8 weeks after femoral surgery. The defect, size, shape and location are presented in supplemental Fig. 1. The rats were divided into four groups with 6 defects per group per time point.
Micro-CT (μCT) analysis of osteogenesis in the femoral defect. After harvesting at each time point,
the femurs were removed intact and fixed in 4% freshly prepared formaldehyde for 24 h at 4 °C. The μCT imaging system (μCT50, Scanco Medical, Brüttisellen, Switzerland) was used to evaluate the osteogenesis within the
Scientific Reports | 6:26925 | DOI: 10.1038/srep26925
3
www.nature.com/scientificreports/
Figure 3. Statistical analysis of bone volume/total volume (BV/TV), trabecular thickness and tissue mineral density in the bone defect as assessed by μCT. All data are shown as the mean ± SD. P
